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Abstract

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of an RBP to specific regions of its target RNAs in adherent human cell lines.

In a recent study, we investigated the role of alternative splicing and RBPs in the acquisition of a drug-resistant (DR) phenotype in pancreatic ductal adenocarcinoma cells (PDAC) (Calabretta et al., 2016). We demonstrated that acquisition of the DR-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which is recruited to the PKM pre-mRNA and favors splicing of the oncogenic PKM2 variant. To investigate the recruitment of PTBP1 on PKM pre-mRNA in vivo, we used the UV cross-linking and immunoprecipitation (CLIP) experimental approach modified from Wang et al. (2009) protocol. Herein, we describe a step-by-step protocol to investigate the direct binding of a specific factor to its RNA target(s), which can be extended to most adherent human cell lines.

Place the dish on ice without lid. Irradiate once with 400 mJ/cm2 (see Figure 1).

Note: UV irradiation produces a covalent bound between RNA and protein that are in contact. The energy level to use depends on availability of aromatic acids. This covalent bound allows purification of RBP/RNA complex under stringent condition. The choice of UV irradiation should be set as the minimum irradiation that allows purification of a control RNA.Figure 1. UV cross-linking. UV cross-linking was performed to bind RNA covalently with proteins. During irradiation the dish is kept on ice to minimize heating.

Remove the PBS and add 0.5 ml of lysis buffer into the 100 mm dish. Note: Do not exceed with lysis buffer.

Alternatively, gently scrape off the cells in cold PBS. Centrifuge for 5 min at 300 x g, at 4 °C. Discard the supernatant, snap freeze the pellets in liquid nitrogen and store at -80 °C.

Cell lysis

Gently resuspend the cells in lysis buffer and transfer the suspended cells to a 1.5 ml centrifuge tube and sonicate on ice [Set: Amplitude (%) at 100 and Cycle at 1] (see Figure 2). Note: Cells tend to aggregate in lysis buffer. Sonication is required to completely dissolve cells aggregate. Usually 5 sec is enough.

Figure 2. Cells sonication. Sonication was performed to dissolve cells aggregate. During sonication the tube is kept in ice to minimize protein denaturation.

Collect the supernatant (cell extract), dilute sample to 1 mg/ml in lysis buffer and store in ice. Note: Lysis buffer composition interferes with Bradford assay. No more than 1 µl of cell extract should be added to 1 ml of Bradford solution.

RNA fragmentation and RBP immunoprecipitation
For each IP use 10 µl of Protein G Dynabeads.

Wash Protein G Dynabeads with 500 µl of lysis buffer and mixing the suspension. Place the tube containing the suspension in the magnetic stand and remove the supernatant. Repeat this step 3 times.

Resuspend the Protein G Dynabeads in 100 µl of lysis buffer and add 3-5 µg of specific polyclonal antibody. Use IgG isotype as a negative control.

After incubation place the samples in the magnetic stand, collect supernatant aliquots (100 µl) from each IPs and discard the residual supernatant. Store aliquots on ice. Note: Aliquots are required for evaluation of RNAse I fragmentation (see step D3, and Figure 4).

Wash IPs twice with 1 ml of high salt buffer and 2 times with 1 ml proteinase K buffer.

Resuspend the Dynabeads in 100 µl of proteinase K buffer. Collect an aliquot of 10% of suspension (10 µl) from each IP. Add 10 µl of Laemmli buffer (2x) to the aliquots and boil aliquot for 5-10 min at 100 °C (see step C10a).Note: Aliquots are required for evaluation of RBP IP by Western-blot analysis (see C10a).

Control of RBPs IP
Run an SDS-PAGE following Western blot analysis using aliquots from step C2 (input) and aliquots from step C10 (IP) samples (Figure 3).

Figure 3. Control of RBP immunoprecipitation in HEK293T cells. Western blot analysis of aliquotes (10%) from step C2 (Input) and step C10 (IPs).

RNA is recovered by adding 1 ml Trizol to the samples. Incubate for 5 min at RT.

Add 200 µl of chloroform and mix vigorously.

Centrifuge at 12,000 x g for 15 min.

After centrifugation, collect half of aqueous phase (≈ 250 µl) to avoid DNA contamination. Note: In our experience, treatment of RNA input sample with DNAse is not sufficient to remove DNA contamination. We prevent DNA contamination by collecting only half of the aqueous phase and avoiding to touch the interphase layer during RNA isolation with Trizol reagent.

Resuspend the RNA in 20 µl of nuclease-free water. After quantification at 260 nm, load the samples on 1.5% denaturating agarose gel for evaluation of RNA fragmentation (Figure 4).

Figure 4. Control of RNA fragmentation. Denaturating agarose gel (1.5%) showing RNA fragmentation in presence of different RNAse I dilutions (1:500 corresponds to 2 U/ml of RNase I), and in presence of different extract concentration (0.8 and 2 mg/ml, left and right panel respectively). The optimal RNA fragmentation is indicated (red box).

Reverse transcription (cDNA synthesis)

Resuspend the RNA pellets from steps D1m and D2k in the same volume of nuclease free water. Note: Volume of water depends on the cDNA synthesis kit used.

Heat for 5 min at 55 °C and store on ice.

Perform cDNA synthesis according to manufacturer’s instructions using half volume of RNA samples. Use the other half to perform cDNA synthesis reaction in absence of reverse transcriptase enzyme (negative control). cDNA synthesis has to be performed using random hexamer primers. Note: Negative control is required to evaluate DNA contamination in all samples.

Real-time quantitative PCR (qRT-PCR)

Design pairs of primer located in different regions of the pre-mRNA, including the region embedding the RBP binding site (Figure 5).Notes:

Optimal PCR product length is 100 nt.

If RBP binding site is not known, it is possible to identify the region in which this is embedded by using several primers pair designed to span the entire pre-mRNA.

Perform qRT-PCR according to manufacturer’s instructions.Note: Quantification cycle (Cq) of negative control should be at least 6 cycles less than relative sample (i.e., Cq negative control = 36; Cq sample = 30).

Figure 5. Schematic representation of primers position along the pre-mRNA for evaluation of RBP binding

Data analysis

Binding of RBP is reported as % of Input in different regions of the RNA target, using the comparative ∆Cq method as follow (Figure 6).

To ensure reproducibility, biological experiments are usually performed in replicates (triplicate). We also recommend to perform each CLIP IPs in duplicate (technical duplicates) to increase the reliability of each experiment (Figure 7). Statistical analysis is performed by t-test procedure.

Figure 7. Data analysis of RBP association to RNA target from three biological replicate experiments. Using hypothetical values, data analysis of RBP binding to pre-mRNA from three biological experiments is shown. By performing technical duplicates for each experiment is possible to eliminate value(s) that deviate from the others. In this example, the value highlighted in red clearly deviates from the other five. Thus, it is conceivable to eliminate it from further analysis, as it may result from a mistake.

Recipes

Note: Make sure to prepare all the following solution in DEPC-treated water.

The CLIP method presented herein is a modified protocol from Wang et al. (2009). This method was employed to identify the binding sites of PTBP1 on the PKM pre-mRNA in Calabretta et al. (2016). The research in our laboratory was supported by the Associazione Italiana Ricerca sul Cancro (AIRC; IG18790), by Telethon Foundation (GGP14095) and by Italian Ministry of Health ‘Ricerca Finalizzata 2011’ (GR-2011-02348423) and '5x1000 Anno 2014' to Fondazione Santa Lucia.

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